This invention relates to lipid compounds and their use, for example for the transport of biologically active substances or molecules in cells.
Liposomes are spherical, self-closed structures composed of lipid bilayers which entrap in their interior a portion of the solvent, in which they float. They may consist of one or more concentric membranes, and their size ranges from several nanometers to several dozens of micrometers.
Liposomes are mostly made from amphiphilic molecules which can be characterized by having a hydrophilic (often named the polar head) and a hydrophobic group (nonpolar tail) on the same molecule. In most cases, liposome-forming molecules are not soluble in water. However, under certain circumstances, they form colloidal dispersions.
Liposomes can be large or small and may be composed from one to several hundred of concentric bilayers. With respect to the size and the nature of the layer (lamellae), they can be classified as multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles LUVs).
SUVs have a diameter from 20 to 600 nm and consist of a single lipid bilayer which surrounds the interior aqueous compartment. LUVs have a diameter from 600 to 30000 nm. MLVs vary greatly in size for up to 10000 nm and contain more than one lipid bilayer, therefore they are multi-compartmental in their structure.
Liposomes can be produced in a number of ways. The so-called xe2x80x9cthin-film hydrationxe2x80x9d method results in the formation of heterogeneous dispersions of predominantly MLVs. By using charged lipid compositions rather high fractions of LUVs can be produced. Said dispersions can be further treated (mechanically, electrostatically or chemically) in order to produce solutions of SUVs. Most frequently these methods include extrusion through filters with pores of different diameter, or sonication.
Alternatively, liposomes can be prepared by lyophilization, where the lipid-film is then dissolved in a volatile solvent (for example tert-butyl alcohol), frozen and lyophilized.
A variety of methods for preparing liposomes have been described in the periodical and patent literature: Szoka and Papahadjopoulos in: Ann. Rev. Biophys. Bioeng. 9, 467-508 (1980) as well as U.S. Pat. Nos. 4,229,360, 4,241,046, 4,235,871.
The most important liposome feature is their ability to dissolve, protect and carry hydrophilic or hydrophobic molecules. For negatively charged drugs, including some proteins, positively charged liposomes can be used. Improvements in therapy were observed, despite the known fact that positively charged liposomes can be toxic.
Various DNA transfection methods have been developed in the past twenty years. These methods include the calcium phosphate precipitation method, DEAE-dextran method, electroporation method, microinjection, receptor mediated endocytosis, liposomes and viral vectors. However, most of these methods posess some significant drawbacks: they are either too inefficient, or too toxic, or too complicated and tedious to be effectively adapted to biological and therapeutical protocols both in vitro and in vivo. For instance, the most frequently used in vitro calcium phosphate precipitation method is too inefficient (average transfection frequency of 1 in 104 cells). Electroporation is much more efficient than the calcium phosphate method. However, this method is too aggressive (maximum efficiency is obtained at about 50% of cell death) and, in addition, this method requires a special apparatus. Microinjection is efficient, but it is too tedious and not practical. All these methods cannot be used in vivo.
A receptor mediated endocytosis method involves polylysine as a basic polymer for interacting and packaging of DNA. Polylysine has been modified with different ligands (transferrin, insulin, asialoorosomukoid, or galactose) in order to target modified protein-DNA complexes to cell surface receptors: Wu, G. Y. et al. in: J. Biol. Chem. (1987) 262:4429-4432, Cotten, M. et al. in: Proc. Natl. Acad. Sci. (USA) (1990) 87:4033-4037, Huckett, B. et al. in: Biochem. Pharmacol. (1990) 40:253-263, Plank, C. et al. in: Bioconjugate Chem. (1992), 533-539. The method has been dramatically improved by use of inactivated adenovirus to facilitate exit of DNA from endosomes, see: Wagner, E. et al. in: Proc. Natl. Acad. Sci. (USA) (1992) 89:6099-6103, Christiano, R. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:2122-2126. The major disadvantage of the described approach includes an inherent inability to control the protein conjugation chemistry and to prepare such conjugates in a reproducible fashion.
At present the best transfection efficiencies both in vitro and in vivo are obtained with retrovirus, adenovirus, and some others, see for example: Kerr, W. G. and Mule, J. J. in: J. Leucocyte Biol. (1994) 56:210-214, Hwu, P. and Rosenberg, S. A. in: Cancer Detect. Prevent. (1994) 18:43-50, Rosenfeld, M. A. et al. in: Cell (1992) 68:143-155. Nevertheless, the use of viral vectors poses several considerations including the requirement of extensive cell culture manipulations, low titers for certain virus systems and the cell tropism of the virus. In addition, immune reactivity against viral vectors may cause problems. Most importantly, the safety issues related to the use of viral vectors are not completely resolved to date.
Liposomes have also been used to introduce DNA into cells both in vitro and in vivo. The most successful liposome systems use different cationic lipids like dioleyloxypropyl-trimethylammonium (DOTMA, which forms a reagent in combination with phosphatidylethanolamine (PE)), dioleoyloxypropyl-trimethylammoniummethyl sulfate (DOTAP), dimethylaminoethane-carbamoyl cholesterol, dioctadecylamidoglycylspermine, 2,3-dioleyloxy-N-(2(sperminecarboxamido)ethyl)-N,N-dimethyl-1 propanamine (DOSPA), which in combination with PE forms a reagent, see: Felgner, P. L. in: Proc. Natl. Acad. Sci. (USA) (1987) 84:7413-7417, U.S. Pat. No. 5,208,036, Leventis, R. and Silvius, J. R. in: Biochimica et Biophysica Acta (1990), 124-132, Gao, X. and Huang, L. in: Biochem. Biophys. Res. Commun. (1991) 179:280-285, Behr, J.-P. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6982-6986.
The advantage of using the above mentioned compounds is that the cationic liposome is simply mixed with DNA and added to the cell. Transfection efficiency is usually high when compared to other physical methods of DNA transfer. Besides for delivery of DNA, a specific compound has been used to deliver mRNA and proteins into cultured cells, see: Malone, R. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6077-6081, and Debs, R. et al. in: J. Biol. Chem. (1990) 265, 10189-10193. Some of the above mentioned compounds have been used to transfect reporter or therapeutically utile genes in vivo, see: Nabel, G. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:11307-11311, Zhu, N. et al. in: Science (1993) 261:209-211. Finally, a DNA transfection protocol has been developed that makes use of the cyclic cationic peptide gramicidin S and PE, see: Legendre, J.-Y. and Szoka, F. C. in: Proc. Natl. Acad. Sci. (USA) (1993), 90:893-897. The above mentioned system takes advantage of the DNA binding ability and the membrane destabilization properties of gramicidin S.
The main disadvantage of cationic liposomes includes their relatively high cytotoxicity. In addition, most of the above mentioned compounds are not active or show highly reduced activity in the presence of serum. Most of them need the use of PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion. Studies on the mechanism responsible for transfection using the cationic lipids have not been fully addressed to date. The need exists, therefore, for a less toxic, non-infectious and more efficient delivery of biological molecules into the cytoplasm and nuclei of living cells.
The object of the present invention is to overcome the above mentioned and other drawbacks of the state of the art. According to a first aspect there are lipid compounds to be provided to allow an improvement of the transport of biologically active agents or molecules through membranes and thus in cells or cell organelles.
This object is solved primarily by the compounds according to claim 1. Preferred compounds are claimed in the subclaims 2 to 11. Derivated products and applications of the novel compounds according to the invention are mentioned in claims 12 to 24 (complexes), claims 25 and 26 (methods for the preparation of complexes), claim 27 (liposome), claims 28 to 32 (liposome formulations) and claims 33 to 42 (methods for the transport of substances and agents through membranes). The wording of all the claims is included into the description by reference.
The compounds according to the invention are illustrated by formula I as follows: 
wherein
R1 and R2 are the same or different and are an alkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms;
R3, R4, and R5 are the same or different and are hydrogen, alkyl, or alkylamine, having from 1 to 8 carbon atoms, or an amino acid, an amino acid derivative, a peptide, or a peptide derivative;
W is hydrogen, a carboxyl group, or a side chain group of amino acids, amino acid derivatives, peptides or peptide derivatives;
Y is a linking group having at least one atom other than hydrogen, particularly xe2x80x94COxe2x80x94, xe2x80x94(CH2)mCOxe2x80x94, xe2x80x94(CH2xe2x80x94)m, xe2x80x94(CHOHCH2xe2x80x94)m, wherein m is from 1 to 20, xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SO2xe2x80x94 or xe2x80x94SO2xe2x80x94;
Z is an ester, ether or amide bond;
n is from 1 to 8; and
X is an anion, particularly a pharmaceutically acceptable anion.
The compounds according to the invention comprise lipids (detergents, surfactants) the main characteristics of which are well-known. The compounds comprised by formula I can be present in the form of their optical isomers (R- or S-configuration) or in the form of mixtures thereof.
According to formula I the lipid compounds are prepared in the form of ionic salts. This is due to the fact that per se neutral compounds are present as ions in (aqueous) solution. Of course the invention is also to include the corresponding neutral compounds. The compounds according to formula I can be provided with further charges by appropriate selection of substituents, for example in the substituent W by appropriate selection of the side chain group of an amino acid.
For a better understanding of the claims and description are given the following definitions:
Alkyl refers to a fully saturated branched or unbranched carbon chain radical.
Alkenyl refers to a branched or unbranched unsaturated carbon chain radical having one or more double bonds.
Alkynyl refers to a branched or unbranched unsaturated carbon chain radical having one or more triple bonds.
Amino acids refer to a monomeric unit of a peptide, polypeptide or protein. The twenty protein amino acids (L-isomers) are: alanine (xe2x80x9cAAxe2x80x9d), arginine (xe2x80x9cRxe2x80x9d), asparagine (xe2x80x9cNxe2x80x9d), aspartic acid (xe2x80x9cDxe2x80x9d), cysteine (xe2x80x9cCxe2x80x9d), glutamine (xe2x80x9cQxe2x80x9d), glutamic acid (xe2x80x9cExe2x80x9d), glycine (xe2x80x9cGxe2x80x9d), histidine (xe2x80x9cHxe2x80x9d), isoleucine (xe2x80x9cIxe2x80x9d), leucine (xe2x80x9cLxe2x80x9d), lysine (xe2x80x9cKxe2x80x9d), methionine (xe2x80x9cMxe2x80x9d), phenylalanine (xe2x80x9cFxe2x80x9d), proline (xe2x80x9cPxe2x80x9d), serine (xe2x80x9cSxe2x80x9d), threonine (xe2x80x9cTxe2x80x9d), tryptophane (xe2x80x9cWxe2x80x9d), tyrosine (xe2x80x9cYxe2x80x9d), and valine (xe2x80x9cVxe2x80x9d). The term amino acid, as used herein, also includes analogues of the protein amino acids, D-isomers of the protein amino acids, xcex2-, y- and other amino acids, unnatural amino acids, and their analogues.
Biologically active substance refers to any molecule or mixture or complex of molecules that exerts biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, steroids, vitamins, polyanions, nucleosides, nucleotides, polynucleotides, etc.
Buffers referred to in this disclosure include: xe2x80x9cHepesxe2x80x9d which is N-2-hydroxyethylpiperazine-Nxe2x80x2-2-ethanesulfonic acid and used here as a buffer at about pH 7; xe2x80x9cPBSxe2x80x9d is a phosphate buffer saline, and is 10 mM phosphate and 0.9% wt. NaCl, used here as an isotonic physiological buffer at pH 7.4; xe2x80x9cTransfection bufferxe2x80x9d is 10 mM Hepes and 0.9% wt. NaCl and used here as a buffer at about pH 7.4; xe2x80x9cTrisxe2x80x9d is tris(hydroxymethyl)amino methane, also used here as a buffer at about pH 7.
Cell-targeted complexes or liposomes applied to the cells include further molecules, e. g. on their surfaces, capable of recognizing a component on the surface of said targeted cell. Cell recognition components include: ligands for cell surface receptors, antibodies to cell surface antigens etc.
Charge-masked complexes or liposomes are to be understood as positively charged, comprising a compound of formula I and a bound polymer which covers the surface of the liposome, for example. A polymer can be covalently linked to any lipid forming the liposome, or be adsorbed on their surface.
A complex is defined as the product made by mixing of two or more components. Such a complex is characterized by a non-covalent interaction (ionic, hydrophilic, hydrophobic etc.) between two or more components.
DNA represents deoxyribonucleic acid which may comprise unnatural nucleotides. DNA may be single-stranded or double-stranded.
Drug refers to any prophylactic or therapeutic compound which is used in the prevention, diagnosis, alleviation, treatment, or cure of disease in a human or an animal.
A liposome formulation is a composition of substances including a liposome which includes entrapped material for diagnostic, biological, therapeutic or other use.
An optional co-lipid is to be understood as a compound capable of producing a stable liposome, either alone, or in combination with other lipid components. Examples of optional co-lipids are phospholipid-related materials, such as lecithin, phosphatidylcholine, dioleylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatic acid, cerebrosides, dicetylphosphate, etc., non-phosphorous lipids like steroids and terpenes. Additional non-phosphorous lipids are, e. g. stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, dioctadecylammonium bromide, amphoteric polymers, triethanolamine lauryl sulfate, cationic lipids described before and the like.
A pharmaceutically acceptable ion is an ion which itself is non-toxic.
A polyanion is a polymeric structure, where more than one unit of the polymer bears a negative charge and the net charge of the polymer is negative.
A polycation is a polymeric structure, where more than one unit of the polymer bears a positive charge and the net charge of the polymer is positive.
A polynucleotide is DNA or RNA containing more than one nucleotide. Polynucleotides are intended to include cyclic polynucleotides and unnatural nucleotides, and can be made by chemical methods, or by use of recombinant technology, or by both.
A polypeptide is to be understood as a series of two or more amino acids coupled via covalent linkage.
RNA represents ribonucleic acid which may comprise unnatural nucleotides. RNA may be single-stranded or double-stranded.
Preferably the above described compounds according to formula I comprise Y that is carbonyl, i. e. xe2x80x94COxe2x80x94. This results in a linkage of the peptide bonding type xe2x80x94COxe2x80x94N less than .
With the lipid compounds according to formula I the group Z preferably includes an ester linkage, i. e. the group xe2x80x94Oxe2x80x94COxe2x80x94.
In further preferred embodiments R1 and R2 include an alkyl or alkenyl group of 10 to 20 carbon atoms, preferably 12 to 18 carbon atoms. The groups R1 and R2 are preferably the same. Of the described preferred groups the alkenyl groups are preferred. Accordingly, the groups R1 and R2 comprise, besides palmityl or stearyl groups, preferably the oleyl group or moieties of the linolic or linoleic acid.
In formula I preferably n is 2. Further, the group W preferably is a side chain group of a basic amino acid, in particular a side chain group of lysine or ornithine.
The groups R3, R4 and R5 may be alkyl groups having 1 to 8 carbon atoms, in particular the methyl group. Preferably the three groups R3, R4 and R5 are the same. In a particularly preferred embodiment R3, R4 and R5 are hydrogen.
Generally, all conceivable anions may be used as counterion X, with pharmaceutically acceptable anions being preferred for most applications of the present invention. Preferably X may be a halide anion, in particular a chloride anion.
Preferred compounds according to formula I are given in claim 10, to which particular reference is made. A particularly preferred compound is L-lysine-bis-(O,Oxe2x80x2-cis-9-octadecenoyl-xcex2-hydroxyethyl)amide-dihydrochloride or an optical isomer thereof.
Compounds covered by the definition of formula I and preferred according to the invention can be prepared by preparing the corresponding compound using the following coupling:
amino acid or peptide-----dialkanol amine-----fatty acid or fatty alcohol
Compounds preparable in this manner are particularly adapted to the applications which will be discussed below. Said compounds are characterized in particular by a considerably minor toxicity than so far known lipid compounds used for similar applications.
Besides the described lipid compounds the invention includes complexes which will be prepared by means of said novel compound. The expression xe2x80x9ccomplexxe2x80x9d is to be understood in the sense of the above definition. It is not necessarily a completely formed liposome. However, as the exact mechanisms of the complex formation are not known, the case that primarily liposomes are formed from lipides and these in turn will complex with polyanions, polycations or complexes thereof, is not excluded according to the invention.
The characteristics of the complexes according to the invention are claimed and described in claims 12 to 17, and 18 to 24, respectively. Explicit reference is made to these claims. The complex formation relies essentially on the fact that the compounds according to the invention have a positive charge.
The ratio between lipide compound and the other constituents of the complex, that is in the (binary) polyanion-lipid complexes the ratio polyanion and lipid compound, may be varied considerably upon the desired application. Preferably, in favour of the lipid compound this ratio is larger than 1:1 (considering charge), so that a positive total or netto charge is resulting for the complex to allow an interaction with the negatively charged surface of biological membranes in a simple manner. If necessary a particularly advantageous ratio will be determined experimentally. Thus for example, in a DNA transfection that ratio of DNA and lipid compound will be determined by experiment which results in an optimized expression of the transfected DNA.
Generally the complexes according to the invention can be realized with all molecules having a negative charge. Preferred are such complexes that use a polynucleotide as the polyanion.
In the ternary complexes given in claims 18 to 24 it is preferred that the polycation is a polypeptide. Generally it is possible to use neutral polypeptides for the formation of ternary polynucleotide-polypeptide-lipid complexes which are also to be covered by the present invention.
Surprisingly, during the formation of ternary complexes was found, for example, that by using polypeptides, the transfection of polynucleotides will be increased significantly as compared to the use of polynucleotide-lipid complexes. The peptide sequences mentioned in claim 23, to which explicit reference is made, can be used preferably. The first sequence represents the C-terminal portion of the nucleoprotein of the hepatitis B virus (HBV). Although a final scientific explanation is not available at present, there seems to be a possible correlation of the positive effect of these peptide sequences in the ternary complexes and the comparably high portion of basic amino acids in the sequences.
The described complexes for particular applications can optionally be charge-masked or cell-targeted for interaction with particular cells or cell organelles.
Further the invention comprises the methods of claims 25 and 26 for the preparation of the complexes according to the invention. The preparation and the contacting involved therein are realized by common methods as well-known to those skilled in the art. Thus for example, in the preparation of binary complexes are combined buffer solutions including the polyanion and the lipid compound, respectively. In a corresponding manner is the preparation of ternary complexes, wherein first a polyanion-polycation complex is obtained in a buffer solution and subsequently the buffer solution is combined with a buffer solution containing the lipid compound.
Further the invention comprises liposomes, which are preparable or prepared from at least one of the lipid compounds according to the invention. The preparation of the liposomes is performed in conventional ways well-known in the state of the art.
Further the invention comprises liposome formulations in aqueous solution including at least one biologically active substance (material, molecule etc.) and one lipid component. Therein the lipid component comprises at least one of the lipid compounds according to the invention. Besides the biologically active substance and the lipid component the liposome formulation includes conventional solution constituents of an aqueous solution, which may be a solution in pure water or preferably conventional buffer solutions.
As is given in the wording of claim 28, a single compound of formula I or a mixture of such compounds may be applied as a lipid component. Furthermore it is possible, to use the compound or compounds according to the invention with additional co-lipids as defined above, for example PE, POPE in mixture.
As long as liposome formation is possible, the amount of biologically active substance is generally not critical. Commonly the substance will be present in amount of up to 10% by weight in the liposome formulation. A value of, for example, 0.01% by weight is to be mentioned as a lower limit. A concentration range of 1 to 5% by weight of biologically active substance is preferred.
The compound according to the invention can preferably amount to 1% to 100% of the lipid component. In preferred embodiments of the liposome formulation there will be one or more co-lipids present, wherein such co-lipids amount to 30 to 70% of the lipid component.
In case the biologically active substance is a drug, the amount thereof is conventionally selected according to the desired therapy. Preferably there will be 1 to 5% by weight of the drug present in the liposome formulation. In case of drugs applied as biologically active substance, there can pharmaceutically acceptable excipients be present in the liposome formulation.
As mentioned above with the complexes according to the invention, also the liposomes or liposome formulations according to the invention will optionally be charge-masked or cell-targeted for interaction with certain cells.
Finally, the invention comprises methods for the transfer of polyanions or polycations or for the transfer of biologically active substances in general through biological membranes, in particular for introduction in cells or cell organelles. In this context particular reference is made to claims 33 to 38, and 39 to 42, respectively. For the methods according to the invention can be used either the compounds according to the invention themselves or compositions including such compounds.
During the performance of the method by incubation in vivo there can additional stabilizers be present, like for example polyethylene glycol.
Further details of the method according to the invention will be described below.
The compounds and other parts of the present invention have various advantages. One of the advantages of the compounds described here are that they allow up to 100% inclusion of polyanionic materials in a convenient protocol. On the other hand, the incubation of positively charged complexes with negatively charged cell surfaces results in a rapid and improved uptake, in particular of polyanionic substances and other biologically active compounds in general. The latter allows the introduction of complexed or included polyanionic substances like for example DNA, in an amount not known so far with such cells.
The particular advantages of the compounds disclosed herein are as follows. First, these compounds represent the novel liposome forming lipids. The geometry of both the aliphatic chains in the compounds of formula I allows their organization in stable double layer structures. The polar head (e. g. amino acid) can be varied as a function of the application. This will allow the easy introduction of different modifications to the amino group, the side chain or the bonding, for example. The cytotoxicity of most of the cationic compounds disclosed here are favourable as compared to that reported with other cationic amphiphiles. All the bondings illustrated in formula I will readily be hydrolized in the cells which results in the formation of non-toxic compounds.
Further, the positively charged lipids of formula I have an improved transfection efficiency in the presence of serum, in contrast to other cationic lipids of the state of the art. The ability to transfect cells in the constant presence of serum is advantageous for various reasons: transfection occurs more easily and is less time consuming, the requirements as to media and serum are reduced, the cells are not depleted of serum which could deteriorate cell functions and viability.
The second specific advantage over the disclosed state of the art is derived from the novel method for the introduction of polynucleotide (in particular DNA) into ternary complexes. Said complex is in particular formed from positively charged lipids of formula I and a complex made of polynucleotide and cationic polypeptide. According to the method there will be formed a first complex made of polynucleotide-cationic polypeptide which in the following step will be complexed with a positively charged lipid of formula I. An exact regulation of the composition will determine the biological activity of the final complex.
The advantage of this procedure in relation to the transfection steps known in the state of the art is for example the fact that the novel method results in an up to 300 times increased transfection efficiency. Furthermore, by using the novel method, a detection of transfection (e. g. detection of the expressed protein) will easily be registered after less than two hours from the start of transfection. Further, the novel procedure allows working in xe2x80x9cmicroscalexe2x80x9d for cell transfection (for example 96-well size) which is desirable for the screening of a large number of samples and allows automation of the process in total.
The compounds of formula I are particularly useful in the preparation of liposomes, however, they can be used in other cases where cationic lipids find application. They may be used in industrial applications, for example. Of particular interest is the application of said compounds in combination with cationic lipids which are acceptable for pharmaceutical formulations (creams, pastes, gels, colloidal dispersions and the like) and/or cosmetic compositions (makeups, lipsticks, polishes, body lotions, moisturizing creams, shampoos and the like).
Formulations comprising the compounds of formula I are advantageous for achieving desirable intracellular delivery of biologically active substances, such as polynucleotides, peptides, proteins, steroids and other natural or synthetic compounds. The intracellular delivery can be into the cytoplasm, into the nucleus, or both. Such intracellular delivery can be achieved in tissue culture (in vitro) and may be used, for example, for transfecting cells with desired polynucleotides (e. g. DNA), or delivery of proteins and the like.
Formulations comprising the compounds of formula I can also be used for ex vivo therapy, where cells isolated from the organism are transfected in vitro and then implanted to the organism. An example of this application is to transfect bone marrow cells.
Intracellular delivery can also be achieved in the whole organism (in vivo) and thus may be useful in several applications like gene therapy, antisense and antigene therapy. Intracellular delivery in vivo can also be used for DNA vaccination with an aim to induce immune response (humoral and/or cellular) to the desired protein.
Intracellular delivery utilizing compounds of formula I is also useful for delivery of anticancer and antiviral compounds, antibiotics and the like.
Cell selectivity can be achieved by incorporating cell recognition compounds, for example on the surface of the vesicle such as antibodies, ligands for cell-surface receptors and the like. Increased stability and further selectivity can be achieved by coating the liposome vesicle with an appropriate charge-masked natural or synthetic compound such as polymers and neutral, or negatively charged lipids.
Liposome vesicles comprising compounds of formula I can be used for the induction of a specific immune response to an antigene of interest which is incorporated in the liposome. Additional components, like N-palmitoyl-S-(2,3-bis(palmitoyl-oxy)-(2RS)-propyl)-R-cystein (Pam3Cys) or N-acetyl-myramyl-L-threonyl-D-isoglutamine (MDP) and derivatives thereof, may be particularly useful.
Compounds of formula I are of interest for the introduction of a lipophilic moiety into polymers, and particulary in peptides in order to increase their uptake by a cell, or to increase their incorporation in the lipid-containing vesicles and the like. Activated compounds of formula I can also be used for modification of proteins.
Further details of the so far presented parts of the invention will be apparent from the following description of preparation procedures, lipid compounds and examples for application thereof. In the description of each example reference will be made to the accompanying drawings.
Method for the Preparation of Selected Compounds of Formula I
Using well-known techniques, a person skilled in the art can readily make additions, deletions or substitutions, increase or decrease the preferred polypeptide amino acid sequences, or simply use another sequence encoding for different cationic polypeptides. It should be understood, however, that such variations are within the scope of this invention. It should also be understood that the examples provided below are for illustrative purposes only and are not to be construed as limiting this invention in any way. 
This reaction scheme is applicable to the sythesis of preferred compounds of formula I, wherein Z is an ester bond and n is 2. In this reaction scheme P1 and P2 are protecting groups, and are the same or different; W is a side chain of amino acid, R1 and R2 are the same and are alkyl or alkenyl having 6 to 24 carbon atoms.
Compounds of formula (A) are commercially available in the optically pure form. To effect the formation of compounds of formula (B), the carboxyl group of the amino acid of formula (A) is activated with an appropriate agent, and is then allowed to react with the imino group of diethanol amine. Methods for activation of amino acids are known in the art. For example, dicyclohexylcarbodiimide/N-hydroxysuccinimide method can be used for epimerization-free amidation of protected amino acids.
The amide of formula (B) is prepared by dissolving the amino acid of formula (A) and a preformed salt of N-hydroxysuccinimide and diethanolamine in an appropriate polar solvent such as dimethylformamide. The mixture is cooled to 0xc2x0 C. and then a solution of dicyclohexylcarbodiimide is added. This reaction is effected by stirring the solution for one hour at 0xc2x0 C. and eight hours at room temperature. The resulting amide of formula (B) is then recovered by some standard separatory means.
To effect the formation of compounds of formula (C), the amide of formula (B) is dissolved in an appropriate solvent (e. g. dichloromethane). To this is added an appropriate tertiary amine, such as, for example, triethylamine in an excess molar amount. The mixture is cooled to about 0xc2x0 C. The alkylating agent of the desired chain length and degree of unsaturation is added in an excess molar amount, preferably about 3 to about 4 times the amount of the amide of formula (B). For example, oleoylchloride can be used to effect the addition of 9-octadecenoyl groups. This mixture is then stirred for approximately 3 hours under N2 atmosphere at room temperature. The product of formula (C) is then extracted. The compound of formula (C) is then deprotected in an appropriate way, depending on the used protecting groups, extracted and further purified by chromatographic means.